Project Abstract Circadian clocks drive the 24-hour oscillations in gene expression and behavior that allow an organism to anticipate the day/night cycle, and its disruption has been implicated in the pathogenesis of cancer, metabolic syndrome, and cardiovascular disease. However, our understanding of conditions where the clock fails remains poor, making it crucial to understand how clocks keep accurate timing over a range of physiological conditions. To function effectively, the clock must maintain a consistent period despite being composed of stochastic biochemical reactions. How does the cell achieve noise-free behavior despite being composed of noisy building blocks, and does molecular noise stemming from limited molecular copy number in the cell impose a significant physical constraint on clock robustness? To answer these questions, I will utilize the circadian clock in the model cyanobacterium Synechococcus elongatus, in which core clock behavior is generated post-translationally entirely by three proteins, KaiA, KaiB, and KaiC, allowing for a direct investigation of the effect of molecular noise on clock function by manipulating Kai protein copy numbers in vivo. The central hypothesis of this proposal is that molecular noise constitutes an important physical constraint on the biochemical robustness of circadian clocks, and that reduction of Kai protein copy number will lead to a loss in accuracy of clock timing that ultimately imposes a fitness penalty on the cell. I have engineered a strain of S. elongatus in which Kai protein copy number is tunable, and I have demonstrated with time lapse microscopy that clock noise increases as Kai copy number is reduced. In my first aim, I will investigate the possibility of cell division as a mechanism explaining the observed increase in noise. In my second aim, I will develop a stochastic model of the Kai system that can predict oscillatory performance as a function of Kai protein copy number. Lastly, by measuring single-cell growth rates on the microscope, I will assess whether noisy rhythms induced by low Kai copy number reduce cellular fitness due to more frequent mistiming between the cell and the environment. These three approaches together will elucidate basic principles of the relationship between noise suppression and molecular copy numbers within the cell.